In vitro nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to attempts to develop them for diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids as in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR; R. K. Saiki, et al. 1985. Science 230, 1350-1354), ligase chain reaction (LCR; D. Y. Wu, et al. 1989. Genomics 4, 560-569; K. Barringer, et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. USA 88, 189-193), transcription-based amplification (D. Y. Kwoh, et at. 1989. Proc. Natl. Acad Sci. USA 86, 1173-1177) and restriction amplification (U.S. Pat. No. 5,102,784) require temperature cycling. In contrast, methods such as Strand Displacement Amplification (SDA; G. T. Walker, et at. 1992. Proc. Natl. Acad. Sci. USA 89, 392-396 and G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696, and EP 0 497 272, all three disclosures being incorporated herein by reference), self-sustained sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad. Sci. USA 87, 1874-1878) and the Q.beta. replicase system (P. M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202) are isothermal reactions. In addition, WO 90/10064 and WO 91/03573 describe use of the bacteriophage phi29 replication origin for isothermal replication of nucleic acids. WO 92/05287 describes a method for isothermal production of sequence-specific oligonucleotides in which a modification in one strand allows a cutting agent to selectively cleave the opposite strand. The single stranded complementary oligonucleotide is released, allowing repolymerization of an additional complementary oligonucleotide. Isothermal amplifications are conducted at a constant temperature, in contrast to the cycling between high and low temperatures characteristic of amplification reactions such as the PCR.
The conventional SDA reaction is conducted at a constant temperature between about 37.degree. C. and 42.degree. C. This is because the exo.sup.- klenow DNA polymerase and particularly the restriction endonuclease (e.g., HincII) are thermolabile (temperature sensitive). The enzymes which drive the amplification are therefore inactivated as the reaction temperature is increased. However, the ability to conduct isothermal amplification reactions such as SDA at higher temperatures than previously possible could have several advantages. Amplification at elevated temperatures may allow for more stringent annealing between amplification primers and template DNA, thereby improving the specificity of the amplification process. Background reactions could also be reduced as a result of such improved amplification specificity. In SDA, a significant source of background reactions are the short "primer dimers" which are generated when the amplification primers interact with each other. Formation of primer dimers may seriously impair the efficiency of the desired, specific amplification of the target sequence. The formation of such primer dimers is more likely at lower temperatures because the reduced stringency of the reaction allows increased transient hybridization between sequences with limited homology. The ability to conduct SDA at higher temperatures could potentially reduce primer dimer interactions, reduce background and improve the efficiency of specific target amplification. In addition, amplifying at higher temperatures may facilitate strand displacement by the polymerase. Improved strand displacing activity might increase the efficiency of target amplification and result in increased yields of the amplification product. The use of sufficiently heat stable enzymes could also allow all reagents required for the SDA reaction to be added prior to the initial heat denaturation step. Conventional SDA requires that the enzymes be added to the reaction mix after double stranded target sequences have been denatured by heating.
dUTP may be incorporated into amplified target DNA by SDA. This allows amplicons from a prior amplification which may contaminate a subsequent amplification reaction to be rendered unamplifiable by treatment with uracil DNA glycosylase (UDG). The decontamination method itself can be used regardless of the temperature at which the amplicons were generated in the SDA reaction. However, SDA amplification products generated at lower temperatures (i.e., 37.degree. C. to 42.degree. C.) may contain a high level of nonspecific background products. Decontamination of large amounts of background amplicons may seriously impede or inhibit elimination of contaminating target-specific amplicons, thus reducing the efficiency of the decontamination procedure. The ability to perform SDA at higher temperatures, by depressing the amount of non-specific background amplicons generated, could therefore increase the efficiency of the UDG decontamination procedure.
The SDA reaction requires several very specific enzymatic activities in order to successfully amplify a target sequence. Thermophilic polymerases have been reported extensively in the literature. However, as other nucleic acid amplification systems do not require the combination of enzymatic activities of SDA, prior to the present invention little was known about the activities and reaction requirements of thermophilic enzymes as they relate to the biological activities required by SDA. Further, because SDA requires concurrent activity by two different enzymes (restriction endonuclease and polymerase), it was not known prior to the present invention whether or not compatible pairs of such thermophilic enzymes existed. That is, both a thermophilic polymerase and a thermophilic restriction endonuclease are required. These two enzymes must have temperature and reaction condition (e.g., salt) requirements compatible with each other and with SDA in order for both to function efficiently in the same SDA reaction mix. In addition, the polymerase must 1) lack 5'-3' exonuclease activity, either naturally or by inactivation, 2) incorporate the modified nucleotides required by SDA (.alpha.thio-dNTPs or other modified dNTPs), 3) displace a downsteam single strand from a double stranded molecule starting at a single stranded nick and preferably 4) incorporate dUTP to allow amplicon decontamination. The polymerase must extend the complementary strand on the template by addition of dNTPs to a free 3'-OH. It is also preferable that the polymerase have a high processivity. That is, the polymerase should be able to add as many nucleotides as possible before dissociating and terminating the extension product. The restriction endonuclease must 1) nick (i.e., cleave a single strand of) its double stranded recognition/cleavage site when the recognition/cleavage site is hemimodified, 2) dissociate from its recognition/cleavage site rapidly enough to allow the polymerase to bind and amplify the target efficiently, and preferably 3) be unaffected by dUTP incorporated into its recognition/cleavage site. In addition, the restriction endonuclease must exhibit these activities under temperature and reaction conditions compatible with the polymerase,